aminoacids and proteins by sunil shah (the great)

1. SUNIL SHAH
BOND KING
AMINO ACIDS AND PROTEINS

3. BASIC STRUCTURES
 An amino acid contains:
 a central carbon bonded to a carboxyl group (-COOH),
 an amino group (NH2), an
 R-group, and
 a hydrogen.
 The R-group is what varies between the 20 amino acids and
gives them unique characteristics.
 The covalent bonds between amino acids are called peptide
bonds.
 It is a bond between the carboxyl group of one amino acid
and the amino group of another amino acid.

4. Abbre
Full Name Side chain type Remarks
v.
Very abundant and versatile. It behaves fairly
neutrally and can be located in both hydrophilic
A Ala Alanine hydrophobic
regions on the outside of the protein and
hydrophobic interior areas.
The sulfur atom binds readily to heavy metal ions.
Under oxidizing conditions, two cysteines can be
joined together by a disulfide bond to form the amino
acid cystine. When cystines are components of a
protein, they enforce tertiary structure and makes
hydrophobic (Nagano, the protein more resistant to unfolding and
C Cys Cysteine
1999) denaturation; disulfide bridges are therefore
common in proteins that have to function in harsh
environments, digestive enzymes (e.g., pepsin and
chymotrypsin), structural proteins (e.g., keratin), and
proteins too small to hold their shape on their own
(eg. insulin).
Behaves similarly to glutamic acid. Carries a
hydrophilic acidic group with strong negative charge.
Aspartic Usually is located on the outer surface of the protein,
D Asp acidic
acid making it water-soluble. Binds to positively-charged
molecules and ions, often used in enzymes to fix the

5. Side chain
Abbrev. Full Name Remarks
type
Phenylalanine, tyrosine, and tryptophan contain large rigid
aromatic group on the side chain. These are the biggest
Phenylala
*F Phe hydrophobic amino acids. Like isoleucine, leucine and valine, they are
nine
hydrophobic and tend to orient towards the interior of the
folded protein molecule.
Because of the two hydrogen atoms at the α carbon, glycine is
not optically active. It is the smallest amino acid, rotates easily,
G Gly Glycine hydrophobic
and adds flexibility to the protein chain. It is able to fit into the
tightest spaces (e.g., the triple helix of collagen).
In even slightly acidic conditions, protonation of the nitrogen
occurs, changing the properties of histidine and the
polypeptide as a whole. It is used by many proteins as a
*H His Histidine basic regulatory mechanism, changing the conformation and
behavior of the polypeptide in acidic regions such as the late
endosome or lysosome, enforcing conformation change in
enzymes.
Isoleucine, leucine and valine have large aliphatic hydrophobic
side chains. Their molecules are rigid, and their mutual
hydrophobic interactions are important for the correct folding
*I Ile Isoleucine hydrophobic
of proteins, as these chains tend to be located inside of the
protein molecule.

6. Side chain
Abbrev. Full Name Remarks
type
Behaves similarly to arginine. Contains a long flexible side-
chain with a positively-charged end. The flexibility of the chain
makes lysine and arginine suitable for binding to molecules
with many negative charges on their surfaces. (e.g., DNA-
*K Lys Lysine basic
binding proteins have their active regions rich with arginine
and lysine.) The strong charge makes these two amino acids
prone to be located on the outer hydrophilic surfaces of the
proteins.
*L Leu Leucine hydrophobic Behaves similar to isoleucine and valine.
Always the first amino acid to be incorporated into a protein;
sometimes removed after translation. Like cysteine, it
contains sulfur, but with a methyl group instead of hydrogen.
*M Met Methionine hydrophobic
This methyl group can be activated, and is used in many
reactions where a new carbon atom is being added to
another molecule.
N Asn Asparagine hydrophilic Neutralized version of aspartic acid.

7. Side chain
Abbrev. Full Name Remarks
type
Contains an unusual ring to the N-end amine group, which
forces the CO-NH amide sequence into a fixed conformation.
Can disrupt protein folding structures like α helix or β sheet,
P Pro Proline hydrophobic
forcing the desired kink in the protein chain. Common in
collagen, where it undergoes a posttranslational modification
to hydroxyproline. Uncommon elsewhere.
Neutralized version of glutamic acid. Used in proteins and as
Q Gln Glutamine hydrophilic
a storage for ammonia.
*R Arg Arginine Basic Functionally similar to lysine.
Serine and threonine have a short group ended with a
hydroxyl group. Its hydrogen is easy to remove, so serine
S Ser Serine Hydrophilic and threonine often act as hydrogen donors in enzymes. Both
are very hydrophilic, therefore the outer regions of soluble
proteins tend to be rich with them.

8. Side chain
Abbrev. Full Name Remarks
type
*T Thr Threonine hydrophilic Behaves similarly to serine.
Behaves similarly to isoleucine and leucine.
*V Val Valine hydrophobic
See isoleucine.
Behaves similarly to phenylalanine and
*W Trp Tryptophan hydrophobic tyrosine (see phenylalanine). Precursor of
serotonin.
Behaves similarly to phenylalanine and
Y Tyr Tyrosine hydrophobic tryptophan (see phenylalanine). Precursor of
melanin, epinephrine, and thyroid hormones.

9. GENERALITIES
Classification  Neutral
• Neutral • Aliphatic
• Aromatic
• Acidic
• Heterocyclic
• Basic • S-containing AA

10. GENERALITIES - NEUTRAL
A. Aliphatic  Hydroxy Amino Acids – they contain
OH group in the side chains
1. Glycine (Gly) (G) – alpha–
amino acetic acid  6. Serine (Ser) (S) or alpha – amino
β – hydroxy propionic acid
2. Alanine (Ala) (A) – alpha –  7. Threonine (Thr) (T) or alpha –
amino propionic acid amino β – hydroxybutyric acid
3. Valine (Val) (V) – alpha–
amino – iso – valeric acid
4. Leucine (Leu) (L) – alpha –
amino – iso – caproic acid
5. Isoleucine (Ile) (l) – alpha –
amino – B – methyl valeric
acid
 Above = Simple monoamino
monocarboxylic acids

11. GENERALITIES
 B. Aromatic  C. Heterocyclic AA
 8. Phenylalanine (Phe) (F)  10. Tryptophan (Trp) (W) –
or alpha – amino – β – alpha – amino β – 3 – indole
phenyl propionic acid propionic
 - often considered
 9. Tyrosine (Tyr) (y) or as aromatic AA since it has
parahydroxy aromatic ring in its structure.
phenylalanine or alpha –  11. Histidine (His) (H) –
amino β – parahydroxy alpha – amino – β -
phenylpropionic acid imidazole propionic acid
 Histidine is basic in solution
on account of the imidazole
ring and often considered as
Basic AA

12. GENERALITIES
 D. Imino Acids  E. „S‟ containing Amino Acids
 12. Proline (Pro) (P) or  - contains 2 sulfur containing
AA
Pyrrolidone – 2 – carboxylic
Acid
 14. Cysteine (Cys) (C) or alpha –
 13. Hydroxyproline (Hyp) or 4 – amino – β – mercaptopropionic
Hydroxy pyrrolidone – 2 – acid
carboxylic acid  S – S linkage is called a
Disulfide bridge
 Proline & Hydroxyproline do  15. Methionine (Met) (M) or alpha
not have a free _NH2 group but – amino y – methylthio - - butyric
only a basic pyrrolidone ring in acid
which the Nitrogen of the Imino
group is in ring but can still
function in the formation of
peptides.
 Are called Imino Acids.

13. GENERALITIES - ACIDIC
 II. Acidic AA
 - AA with 2 _COOH groups and 1 _NH2 group
 - monoaminodicarboxylic Acids
 16. Aspartic Acid (Asp) (P) or alpha – amino succinic acid
 Asparagine (Asn) (N) or delta amide of alpha – amino
succinic acid
 17. Glutamic Acid (Glu) (E) or alpha aminoglutaric acid
 Glutamine (Gln) (Q) – amide of glutamic acid or 8 –
amide of alpha – amino glutaric acid

14. GENERALITIES - BASIC
 III. Basic AA
 - AA with 1 – COOH group and 2 – NH2
groups
 - Diamino monocarboxylic acids
 18. Arginine (Arg) (R) or alpha – amino – 8 – guanidino
- valeric acid
 19. Lysine (Lys) (K) or alpha – E diamino
 8 – hydroxy - - valeric acid
 As already mentioned Histidine – is also classified as
Basic AA

15. GENERALITIES
 Classificationof Amino Acids
based on polarity
 of the R group:
• 4 groups
• Polarity  reflects the
functional role
of AA in protein structure

16. GENERALITIES
1. Non-polar AA
• hydrophobic (water hating)
• No charge on the ‘R’ group
• Examples are:
Alanine Methionine
Leucine
Phenylalanine
Isoleucine
Tryptophan

17. GENERALITIES
 2. Polar AA with (+) ‘R’ group
• carries (+) charge
• Examples:
Histidine Arginine
Lysine
 3. Polar AA with (-) ‘R’ group
• carries (-) charge
• Examples:
Glutamic Acid
Aspartic Acid

18. GENERALITIES
 4. Polar AA with no charge
 on ‘R’ group
• no charge on the ‘R’ group
• possess groups  hydroxyl
sulfhydryl
amide
• participate in hydrogen bonding of
• protein structure
• Examples:
Asparagine Glycine Cysteine
Tyrosine Serine Threonine

19. GENERALITIES
 Zwitterion or dipolar ion:
 Zwitter
  from German word – means
“hybrid”
 Zwitter ion (or dipolar ion)
  a hybrid molecule containing
(+) and (-) ionic groups

20. GENERALITIES
AA rarely exist in a neutral form with free
carboxylic (-COOH) and free Amino (-NH2)
groups
Strongly acidic pH (low pH)  AA (+)
charged (cation)
Strongly alkaline pH (high pH)  AA (-)
charged (anion)
Each AA has a characteristic pH (e.g. Leucine,
pH – 6.0), at which it carries both (+) and (-)
charges and exist as zwitterion

21. GENERALITIES
 Isoelectric pH (symbol pI)
the pH at which a molecule exists as a
Zwitterion or dipolar ion and carries
no net charge
Molecule is electrically neutral

22. GENERALITIES
Tryptophan
Histidine Arginine Leucine
Valine
Phenylalanine PVT TIM HALL Lysine
Threonine Methionine
Isoleucine
Essential Amino Acids

23. Essential Amino Acids (Body cannot make these amino acids, they must come
from food or amino acid supplements.)
Min.
Dail Acid
y Base # of pathways - One Generate Works
Amino Acid Sym Abbr mg Neu. Pathway s With Augments
1 - threonine -->
1 Isoleucine* I Ile 10 n - - muscles
isoleucine
blood,
1 - keto-isovalerate --
2 Leucine* L Leu 14 n - - muscle,
> leucine
hormone
herpes,
3 Lysine K Lys 12 B 2 - asparate --> lysine - calcium
triglycerides
7 - homoserine --> seleniu hair, skin,
4 Methionine M Met 13 n cysteine
methionine m, zinc chelator
Phenylalanin 3 - chorismate -->
5 F Phe 14 n tyrosine B6 depression
e phenylalanine
collagen,
3 - aspartate --> glycine,
6 Threonine T Thr 7 N - tooth
threonine serine
enamel
1 - chorismate --> niacin,
7 Tryptophan W Trp 3.5 n - depression
tryptophan seratonin
8 Valine* V Val 10 n 1 - pyruvate --> valine - - muscles
Food Sources: Fish - meat - poultry - cottage cheese - peanuts - lentils
A= acid, B= base, N= Neutral, n= non-polar

24. Non-Essential Amino Acids (The body can make these amino acids from the above essential amino
acids.)
Min. Acid
Daily Base # of pathways - One
Amino Acid Sym. Abbr mg. Neu. Pathway Generates Works With Augments
4 - valine pyruvate -->
1 Alanine A Ala - n - - -
alanine
immune,
polyamines,
2 Arginine* R Arg - B 4 - citrulline --> arginine zinc (lysine) healing,
creatine
muscles
3 Asparagine N Asn - N 3 - asparate and ammonia - - CNS
Aspartic
4 D Asp - A 3 - glutamate --> aspartate - - CNS, brain
Acid
homocysteine
5 Cysteine C Cys - N 4 - serine --> cysteine B6, Vit.E skin, hair
, taurine
Glutamic 3 - ketoglutarate --> glutamine,
6 E Glu - A B6 brain
Acid glutamate GABA
7 Glutamine Q Gln - N 2 - glutamate --> glutamine - - brain
GABA,
8 Glycine G Gly - N 2 - serine --> glycine glutathione body protein
taurine
blood, allergy,
9 Histidine* H His - B 1 - histidinol --> histidine histamine -
sex
hydroxyprolin collagen,
10 Proline P Pro - n 4 - l-glutamine --> proline Vitamin C
e elastin
1 - phosphoglycerate --> cysteine,
11 Serine S Ser - N choline blood sugar
serine glycine
2 - phenylalanine --> thyroxin,
12 Tyrosine Y Tyr - N B6 thyroid
tyrosine melanin
* = These are Essential for infants, since their bodies cannot produce them yet.

25. Other Amino Acid Factors
Lysine,
carnitine --> fat
- Carnitine - Car - - methionin Vitamin C
butyrobetaine --> metabolism
e
argenine --> citrulline
- Citrulline - Cit - - Ornithine zinc urea cycle
--> ornithine
Hydroxy Hy proline -->
- - - - - Vitamin C collagen
proline p hydroxyproline
Or argenine --> citrulline
- Ornithine - - - - - urea cycle
n --> ornithine
methionine --> GABA,
- Taurine - Tau - - B6 heart, bile
cysteine --> taurine glycine
The above are precursors for, or important products of, the 20 "true" amino acids
listed above.

26. AMINO ACID SYNTHESIS
 ALL ARE SYNTHESIZED FROM COMMON
METABOLIC INTERMEDIATES
 NON-ESSENTIAL
 Transamination of -KETOACIDS that are
available as common intermediates
 All except tyrosine are derived from one of the
following common intermediates: pyruvate,
oxalacetate, -KG, 3-phosphoglycerate
 ESSENTIAL
 Their -KETOACIDS are not common
intermediates (Enzymes needed to form them
are lacking)

27. Amino Acids Glucogenic Glucogenic and Ketogenic
A
Ketogenic
m Non-Essential Alanine Tyroxine
i AA Asparagine
n
o Aspartate
A Cysteine
c Glutamate
i
d Glutamine
M Glycine
e Proline
t
a
Serine
b
o Essential AA Arginine Isoleucine Leucine
l
Histidine Phenylalanine Lysine
i
s Methionine Tryptophan
m Threonine
Valine
Glucogenic – amino acids that give rise to pyruvate and
citric acid cycle intermediates that can be turned into
glucose
Ketogenic – amino acids that give rise to acetoacetate and

28. AMINOACIDOPATHIES

29. TYROSINEMIA
 DEFECTIVE PROCESS: AMINO ACID TRANSPORT – EXCRETION
OF TYROSINE AND TRYPTOPHAN CATABOLITES IN URINE
 DEFECTIVE ENZYME:
• TYROSINE AMINOTRANSFERASE (II);
• 4-HYDROXY-PHENYLPYRUVATE OXIDASE (III);
• FUMARYLACETOACETATE HYDROLASE (I)

30. ALKAPTONURIA
 DEFECTIVE PROCESS: TYROSINE
DEGRADATION
 DEFECTIVE ENZYME: HOMOGENTISATE-
1,2-DIOXYGENASE
• Original “inborn error of metabolism
• Darkening of urine at long standing
• OCHRONOSIS – generalized pigmentation of
tissues and arthritislike degeneration

31. PHENYLKETONURIA
 DEFECTIVE PROCESS: CONVERSION OF
PHENYLALANINE TO TYROSINE
 DEFECTIVE ENZYME: PHENYLALANINE
HYDROXYLASE (Phenylalanine-4-mono-oxygenase)
 An autosomal recessive trait – urine has a musty
odor
 Compounds seen in both urine and blood:
 Phenylpyruvic acid – primary catabolite
 Phenyllactic acid – product of deaminzation
 Phenylacetylglutamine – produced from oxidation of
phenylpyruvic acid

32. PHENYLKETONURIA
 NOT RESPONSIVE TO DIET: DEFICIENCY OF ENZYME S NEEDED FOR
THE REGENERATION AND SYNTHESIS OF TETRAHYDROBIOPTERIN
(BH4)
• High phenylalanine and deficiency of production of neurotransmitters
from tyrosine and tryptophan
• Administration of L-dopa and 5-OH tryptophan
 SCREENING: GUTHRIE BACTERIAL INHIBITION ASSAY – B. subtilis +
β2-thienylalanine
• Semi-qualitative method: phenylalanine >2-4 mg/dL will result to
bacterial growth indicative of a POS (+) result
• Microfluorometric assay – based on the fluorescence complex formed of
phenylalanine-ninhydrin copper in the presence of dipeptides (L-leucyl-L-
alanine)
• Quantitative method: filter paper is pretreated with trichloroacetic acid
added with ninhydrin, succinate and leucylalanine in the presence of
copper tartrate and read at excitation and emission wavelengths of
360nm and 530nm
 HPLC – reference method (1.2 -3.4 mg/dL)

33. MAPLE SYRUP DISEASE
 DEFECTIVE PROCESS: METABOLISM OF THREE ESSENTIAL
BRANCHED-CHAINAMINO ACIDS (LEUCINE, ISOLEUCINE AND VALINE)
 DEFECTIVE ENZYME: BRANCHED CHAIN α-KETO ACID
DECARBOXYLASE COMPLEX
• Burnt sugar odor of urine, breath and skin
• Screening: Modified Guthrie test – metabolic inhibitor of B. subtilis which is
4-azaleucine is impregnated in the medium
• POS (+) for MSUD = bacterial growth
• Microfluorometric assay using leucine dehydrogenase
• Filter paper specimen is treated with methanol and acetone to denature
hemoglobin
• The NADH fluorescence produced is measured at 450nm; excitation
wavelength at 360nm
• Confirmed diagnosis is based on finding increased plasma and urinary levels
of the three branched-chain amino acids and their ketoacids with LEUCINE
(highest: above 4mg/dL)
• Prenatal diagnosis: measuring decarboxylase enzyme concentration in cells
cultured from amniotic fluid

34. CYSTINURIA
 DEFECTIVE PROCESS: AMINO ACID TRANSPORT
SYSTEM RATHER THAN A METABOLIC ENZYME
DEFICIENCY
• 20 – 30 fold increase in the urinary excretion of cystine
as a result of genetic defect in the renal resorptive
mechanism
• Other amino acids excreted: ornithine, lysine and
arginine
• Cystine being relatively insoluble and once
accumulated will tend to precipitate in the kidney
tubules forming calculi
• Remedy: high fluid intake and alkalinizing the urine:
penicillin
• Diagnosis: Cyanide-nitroprusside (red-purple color)

35. ARGININOSUCCINIC ACIDURIA AND
CITRULLINEMIA PROCESS: UREA CYCLE
 DEFECTIVE
 DEFECTIVE ENZYME: ARGININOSUCCINIC
ACID LYASE
• Decrease in activity of ASA synthetase 
Citrullinemia
• Citrulline is elevated in MS/MS
• Argininosuccinic aciduria – milder
elevations
• Citrullinemia – dramatic elevations
• * Ornithine and arginine are seen
increased in older infants

36. ISOVALERIC ACIDEMIA
DEFECTIVE PROCESS: DEGRATIVE
PATHWAY OF LEUCINE
DEFICIENCY ENZYME: ISOVALERYL-CoA
DEHYDROGENASE
• “Sweaty feet” odor urine
• Elevations of glycine conjugate: isovaleric
acid and isovalerylglycine

38. GENERAL CHARACTERISTICS
 Structure of Proteins
• PRIMARY
• SECONDARY
• TERTIARY
• QUARTERNARY

39. FUNCTIONS OF PROTEINS
 1. Enzymatic catalysis
 2. Transport and storage
 3. Coordinated motion
 responsible for movements in the body
(muscles)
 4. Mechanical support
 support of body - bones and muscles
 5. Generation and transmission of impulses
 6. Control of growth and differentiation
 AA - simplest form of proteins
 20 AA

40. CHARACTERISTICS OF PROTEINS
 1. Have more members
 2. Fundamental component of protoplasm
 3. Elements in protein
 C, H, O, N (I & Fe)
 4. Proteins are considered macromolecules,
composed of a number of AA joined together by
peptide bond or linkage
 5. Only foodstuff when absent in the diet will
cause death
 6. Utilize in the body for growth

41. PROTEIN METABOLISM
GASTRIC
• Digestion of protein begins in the stomach
where the enzyme pepsin is secreted by the
chief cells as proenzyme or zymogen
(inactive form)
• Pesinogen – activated by HCL secreted by
the parietal cells
• By autoactivation – pepsin itself stimulates
its own activation

42. PROTEIN METABOLISM
 PANCREATIC
• Once protein reaches the duedenum, exocrine
pancreatic secretion: trypsin,chymotrypsin,
elastase and carboxypeptidase in their inactive
forms are release
• Endopeptidase – cleave protein in the
internal sites
• Exopeptidase – cleave one amino acid
from the carboxyl terminus of the
polypeptide
Secretin stimulates the pancreas to
produce a protein-free electrolyte solution
rich in bicarbonates

43. PROTEIN METABOLISM
 INTESTINAL
• This mediated by peptidase produced by the
mucosal cells
• Amino peptidases and dipeptidases hydrolyze the
residual peptides
• The end products of protein digestion are amino
acids and some short peptides

44. AMINO ACID POOL
 Primarily for the  Also for the synthesis of
synthesis of body nonprotein nitrogen-
proteins: containing compounds
• Plasma • Purines
• Intracellular proteins • Pyrimidines
• Structural proteins • Creatine
• Porphyrins
• Histamines

45. PROTEIN MEASUREMENT
 PLASMA TOTAL  GLOBULIN:
PROTEIN: • 23 – 35 g/L
• 60 – 70 g/L (SI units) • 2.3 – 3.5 g/dL
• 6.0 – 7.0 g/dL
(Conventional unit) FIBRINOGEN:
• 2 – 4 g/L
ALBUMIN:
• 0.2 – 0.4 g/dL
• 32 – 45 g/L
• 3.2 – 4.5 g/dL

46. DETERMINATION OF PROTEIN NITROGEN
 KJELDAHL
• Reference (standard) method for the determination
of protein concentration
• Liberation the nitrogen content from proteins is
measured by oxidation with heat at 350oC and
strong sulfuric acid
• Catalysts used during the digestion: copper sulfate,
mercuric sulfate or selenium oxychloride
• Nitrogen then is converted to ammonium ion
• Separated from the digestant by steam
distillation
• Ammonium ion is liberated and are measured
by titration with an alkali, Nesslerization or

47.  NESSLERIZATION
• Liberation of ammonium ions with Nessler‟s reagent –
double iodide of potassium and mercury  dimercuric
ammonium iodide (yellow to orange brown product
• Colloidal stabilizer: gum ghatti
BERTHELOT
• Idophenol reaction
• Ammonia is allowed to react with alkaline hypochlorite to
form indophenol blue solution
• Catalyst: sodium nitroprusside

48. BIURET REACTION
 Based on the ability of the peptide bonds to react with
copper ions to form purple “biuret” complex
 Biuret reagent contains:
• Copper sulfate – biuret formation
• Sodium hydroxide
• Rochelle salt (potassium sodium tartrate) – stabilizes
the copper sulfate
• Potassium iodide – keeps copper ions in cupric state
• The purple complex is measure at 540 – 560 nm
 Total proteins – measure by allowing to react with the
biuret reagent
 “Salting-out process” – precipitation of globulins with 22-
26% sodium sulfate to get the albumin content

49. FOLIN-CIOCALTEAU METHOD
 based on the ability of phosphotungstomolybdic
acid (phenol reagent or Folin-Ciocalteau‟s reagent)
to oxidize the phenolic structures of the amino
acids tyrosine, phenylalanine, tryptophan and
histidine.

50. LOWRY PROTEIN ASSAY
• Uses biuret method followed by the
Folin-Ciocalteau‟s method
• Color obtained is enhance and provide a
more sensitive method
• Consistently obtained accurate protein
determination

51. COOMASIE BRILLIANT BLUE DYE
• Free from interferences
• It can detect proteins as low as 1 ug in
concentration

52. NINHYDRIN REACTION
• Used to detect peptides and amino acids
after paper chromatography
• Violet color formation upon reaction to
ninhydrin (tri-keto-hydrindine hydrate
and amines
• Results are comparable to Coomasie
dye method

53. REFRACTOMETRY
• Measuring refractive index of serum
• Accurate levels are obtained at protein
concentrations greater than 2.5 g/dL

54. SPECIFIC GRAVITY
• Copper sulfate standards with known
specific gravity
• Measurement of hemoglobin

55. TURBIDIMETRY
• Measures total proteins and globulins
in urine and CSF
• Proteins are precipitated by
sulfosalicylic acids, trichloroacetic
acid, acetic acid-potassium
ferricyanide solution

56. UV ABSORPTION METHOD
• Proteins absorb UV light at 280 nm
• Mostly due to the presence of tryptophan,
phenylalanine and tyrosine
• Quantifies protein in the range of 0.5 to 1.5
mg/dL

57. ELECTROPHORESIS
Sodium dodecyl sulfate polyacrilamide gel
electrophoresis
• Separates protein according to molecular weight
and isoelectric focusing which separates proteins
on the basis of isoelectric poins
• After separation, proteins are quantified by densitometry
(preferred method and by elution and spectrophotometry
Qualitative characterization of proteins may be stained
with appropriate dyes:
1. Coomasie brilliant blue
2. Ponceau S
3. Amido black
4. Silver staining - most sensitive and measures proteins
in nanogram concentration

58. PRECIPITATION
 Precipitants:
• Sodium sulfate
• Ammonium sulfate
• Methanol

59. PLASMA PROTEIN
 PRE-ALBUMIN
• Migrates faster than albumin toward the
anode
• Has the ability to bind with thyroxine (TBPA)
and complex with vitamin A
• Very rich with tryptophan
• Marker of nutritional status
• Crosses the plancenta

60. ALBUMIN
• Single most abundant protein in normal plasma
• About 2/3 of the total plasma proteins
• Regulates intravascular oncotic pressure
• Loss of albumin is seen in ascites, protein
losing nephropathy and protein losing
enteropathy
• Seen in peripheral edema
 Functions of albumin:
1. Regulation of oncotic pressure
2. Amino acid reservoir
3. Transport of small molecules

61. ALBUMIN
• Bounded by thyroxine, bilirubin, penicillin,
cortisol, estrogen and free fatty acids
• Calcium and magnesium
• Analbuminemia – absence of albumin in
the blood
• Bisalbuminemia – two separated albumin
bands after electrophoresis

62. ALPHA1-ANTITRYPSIN
• Major component of the alpha1-globulin
fraction
• Acts as protease inhibitor
• Combines with and inactivates trypsin and
elastase
• In lungs, elastase is released by the
neutrophils during inflammatory conditions
to combat the invading microorganisms
• Deficiency will result to emphysema
• Also an intrinsic factor in homeostatic
mechanism modulating endogenous
proteolysis with the body

63. ALPHA2-MACROGLOBULIN
• one of the largest non-immunoglobulin
proteins in the plasma
• Rises tenfold or more in nephrotic
syndrome when other low molecular
weight proteins are lost
• In nephrotic syndrome, the levels of
alpha2-macroglobulin may be equal to or
greater than that of albumin (2-3 g/dL)

64. HAPTOGLOBIN
• Haptoglobin migrates in the alpha2-region
• Combines with hemoglobin released by lysis of red
blood cells in order to preserve body iron and
protein stores
• Hemoglobin-haptoglobin complex is then taken by
the RES where the hemoglobin fraction is broken
into iron and bilirubin
Hemoglobinuria vs myoglobinuria:
• Peroxidase acitivity using dipstick or chemstrips –
urine specimen
• Increased or normal free haptoglobinuria will
indicate myoglobinuria or rhabdomyolysis

65. BETA-LIPOPROTEIN
 Low-density lipoprotein which has a
characteristic sharp leading edge and a
feathery trailing edge

66. TRANSFERRIN
• Also known as siderophilin
• Major beta-globulin protein
• Normally at 200 to 400 mg/dL
• Transport ferric ions from iron stores of
the intracellular or mucosal ferritin to bone
marrow where the red blood cells
precursors and other cells have
transferrin receptor on their surfaces
• Measured in terms of iron-binding
capacity – seen elevated in anemia

67. COMPLEMENT
• Travels with beta-globulins during the
electrophoresis
• C3 is decreased in autoimmune diseases

68. FIBRINOGEN
• Most abundant of the coagulation factors
• Elevation seen in pregnancies and the
use of birth control pills
• Decreased during the activation of
coagulation factors
• Migrates between beta and gamma
fractions
• Measured by Parfentjev method which
uses ammonium sulfate and sodium
chloride

69. MINOR PROTEINS
• Ceruloplasmin – Wilson‟s disease
(hepatolenticular degeneration)
• Gc-globulin – binds with vit D
• Hemopexin – prevent excretion of heme
and iron
• Alpha1-acid glycoprotein – increased
during pregnancy
• C-reactive protein – seen in tissue
necrosis; most sensitive acute phase
reactant located at chromosome 1